Mechanism and regulation of DNA double-strand break repair

染色体双链断裂(DSB)是在正常细胞过程中自发发生的细胞毒性损伤 用DNA损伤剂对细胞进行新陈代谢或随后的处理。如果未修复或修复不当， DSB可能会导致严重的有害事件，如染色体丢失、缺失、复制或 易位。DSB的修复缺陷会导致基因组的不稳定，表现为免疫学上的， 发育或神经缺陷，以及癌症的易感性。DSB的毒性被开发用于 放射和化疗，以及针对特定DNA修复蛋白的靶向治疗。因此， 了解DSB修复机理具有重要的基础性意义和实际应用价值 开发新的治疗方法并发现抗药性的途径。通常，细胞通过以下两种方式之一修复DSB 同源重组(HR)或非同源末端连接(NHEJ)。人力资源采用了广泛的同源性和 模板DNA合成以修复断裂的染色体，被认为是一个没有错误的过程。 NHEJ直接连接DSB末端，这一机制由于小的删除或 在交叉点处插入。在这两条途径之间的选择由细胞周期决定，细胞周期 调节HR的早期步骤，即5‘-3’切除DSB。我们研究计划的总体目标是 破译依赖同源的DSB修复机制，以酿酒酵母为例 模型系统。我们计划的第一部分建立在我们之前的研究基础上，表明保守的Mre11- RAD50-Xrs2(MRX)复合体启动5‘-3’切除。具体地说，我们将使用下一代测序 确定MRX划痕的部位，确定染色质结构如何影响划痕部位的选择和 测量HR修复过程中细胞的切除束长度。除了控制末端切除之外， MRX促进NHEJ，系住DSB末端，并将Tel1ATM激酶招募到DSB以激活DNA损伤 检查站。我们将使用特定的方法来确定这些不同功能对基因组完整性的贡献 MRX组分的等位基因与测量总染色体重排的分析相耦合。出现的DSB 由于复制叉状塌陷或无帽端粒的侵蚀，端粒只有一个自由端，可通过 链侵入同源双链DNA，然后复制到染色体末端(断裂诱导 复制，BIR)。我们研究计划的第二部分利用了在 我的实验室用BIR研究DNA合成的机制和保真度。

Chromosomal double strand breaks (DSBs) are cytotoxic lesions that occur spontaneously during normal cell metabolism or following treatment of cells with DNA-damaging agents. If unrepaired or repaired inappropriately, DSBs can lead to profoundly detrimental events, such as chromosome loss, deletions, duplications or translocations. Defects in the repair of DSBs cause genomic instability, manifested as immunological, development or neurological defects, and predisposition to cancer. The toxicity of DSBs is exploited for radiation and chemotherapy, as well as targeted therapies directed against specific DNA repair proteins. Thus understanding the mechanisms of DSB repair is of fundamental importance and has practical application for development of new therapeutics and uncovering pathways to resistance. Typically, cells repair DSBs by either homologous recombination (HR) or non-homologous end joining (NHEJ). HR employs extensive homology and templated DNA synthesis to restore the broken chromosome and is considered to be an error-free process. NHEJ directly ligates DSB ends, a mechanism that is potentially error prone due to small deletions or insertions at the junctions. The choice between these two pathways is governed by the cell cycle, which regulates an early step in HR, namely, 5'-3' resection of DSBs. The overall goal of our research program is to decipher the mechanisms of homology-dependent DSB repair, using the yeast Saccharomyces cerevisiae as a model system. The first part of our program builds on our previous studies showing that the conserved Mre11- Rad50-Xrs2 (MRX) complex initiates 5'-3' resection. Specifically, we will use next-generation sequencing to identify the sites of MRX nicking, determine how chromatin structure influences nick site selection and measure the length of resection tracts in cells undergoing HR repair. In addition to controlling end resection, MRX promotes NHEJ, tethers DSB ends and recruits the Tel1ATM kinase to DSBs to activate the DNA damage checkpoint. We will determine the contribution of these diverse functions to genome integrity using specific alleles of MRX components coupled to assays measuring gross chromosome rearrangements. DSBs that arise by replication fork collapse or by erosion of uncapped telomeres have only one free end and are repaired by strand invasion into a homologous duplex DNA followed by replication to the chromosome end (break-induced replication, BIR). The second part of our research program utilizes physical and genetic assays developed in my laboratory to address the mechanism and fidelity of DNA synthesis by BIR.